US20250304671A1 - Stable aqueous formulation of an anti-adrenomedullin (adm) antibody or anti-adm antibody fragment - Google Patents

Stable aqueous formulation of an anti-adrenomedullin (adm) antibody or anti-adm antibody fragment

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US20250304671A1
US20250304671A1 US18/844,746 US202318844746A US2025304671A1 US 20250304671 A1 US20250304671 A1 US 20250304671A1 US 202318844746 A US202318844746 A US 202318844746A US 2025304671 A1 US2025304671 A1 US 2025304671A1
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adm
antibody
adrenomedullin
patient
formulation
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US18/844,746
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English (en)
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Jarrid GOLDSTEIN
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Adrenomed AG
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Adrenomed AG
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Assigned to ADRENOMED AG reassignment ADRENOMED AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GOLDSTEIN, Jarrid
Publication of US20250304671A1 publication Critical patent/US20250304671A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of pharmaceutical formulations of antibodies. Specifically, the subject matter of the present invention relates to a stable liquid antibody formulation comprising Arginine, Trehalose, a surfactant and Histidine and its pharmaceutical preparation and use.
  • ADM anti-adrenomedullin
  • the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates a deviation from the indicated numerical value of ⁇ 20%, preferably ⁇ 15%, more preferably ⁇ 10%, and even more preferably ⁇ 5%.
  • the ADM antibody or an ADM antibody fragment is present in a total amount of at least 5 mg, particularly at least 10 mg, more particularly from 10 mg to 1000 mg, more particularly from 50 mg to 700 mg, more particularly from 100 mg to 500 mg and most particularly from 200 to 480 mg referring to a 10 ml vial.
  • This total amount of the ADM antibody or an ADM antibody fragment can e.g., be used to prepare a ready-to-application solution by diluting said total amount of the ADM antibody or an ADM antibody fragment in a suitable buffer comprising phosphate buffered saline solution, water and emulsions such as oil/water emulsion to a desired concentration.
  • the ADM antibody or an ADM antibody fragment is present in a total amount of at least 5 mg, particularly at least 10 mg, more particularly from 10 mg to 1000 mg, more particularly from 50 mg to 700 mg, more particularly from 100 mg to 500 mg and most particularly from 200 to 480 mg referring to a 10 ml vial and may be diluted in a suitable buffer comprising phosphate buffered saline solution, water and emulsions such as oil/water emulsion to 10 ml to 200 ml, preferably 20 ml to 180 ml, more preferably 30 ml to 150 ml and most preferred to 50 ml to 100 ml.
  • a suitable buffer comprising phosphate buffered saline solution, water and emulsions such as oil/water emulsion to 10 ml to 200 ml, preferably 20 ml to 180 ml, more preferably 30 ml to 150 ml and most preferred to 50 ml to 100 ml.
  • the pharmaceutical aqueous formulation is further diluted in an infusion solution and applied via infusion to the patient.
  • the physician as person skilled in the art would prepare a ready-for-application solution from the pharmaceutical aqueous formulation according to the patient's needs and then directly apply the ready-for-application solution to the patient.
  • the ADM antibody or an ADM antibody fragment may be administered in a dose of at least 0.5 mg/kg body weight, particularly at least 1.0 mg/kg body weight, more particularly, from 1.0 to 20.0 mg/kg body weight, e.g., from 2.0 to 10 mg/kg body weight, from 2.0 to 8.0 mg/kg body weight or from 3.0 to 5.0 mg/kg body weight.
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to the N-terminal region (aa 1-21) of ADM (SEQ ID No. 1) or an antibody fragment thereof wherein the heavy chain comprises the sequences:
  • ADM human or humanized anti-adrenomedullin
  • SEQ ID No. 1 human monoclonal antibody or fragment that binds to the N-terminal region (aa 1-21) of ADM (SEQ ID No. 1) or an antibody fragment thereof wherein the heavy chain comprises the sequences:
  • CDR1 SEQ ID NO: 5 QSIVYSNGNTY
  • CDR2 SEQ ID NO: 6 RVS
  • CDR3 SEQ ID NO: 7 FQGSHIPYT.
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment comprising an Anti-Adrenomedullin antibody directed to the N-terminal end of Adrenomedullin comprising the following sequence as a heavy chain:
  • ADM human or humanized anti-adrenomedullin
  • the identity defines the percentage of amino acids with a direct match in the alignment.
  • Surfactants can be used to alter the surface tension of a liquid antibody formulation.
  • the surfactant reduces the surface tension of a liquid antibody formulation.
  • the surfactant can contribute to an improvement in stability of any of the antibody in the formulation.
  • the surfactant can also reduce aggregation of the formulated antibody (e.g., during shipping and storage) and/or minimize the formation of particulates in the formulation and/or reduces adsorption (e.g., adsorption to a container).
  • the surfactant can also improve stability of the antibody during and after a freeze/thaw cycle.
  • the surfactant can be, for example without limitation, a polysorbate, poloxamer, triton, sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine, isostearamidopropyl-
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said surfactant is Poloxamer.
  • poloxamer is selected from the group comprising copolymers based on ethylene oxide and propylene oxide comprising but not limited to L64, P65, P84, P85, F88, P103, P104, P105, F108, P123 or F127.
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said formulation is essentially free of NaCl and/or Glycine.
  • ADM anti-adrenomedullin
  • the aqueous formulation comprises histidine, wherein the histidine can comprise either L-histidine or D-histidine, a solvated form of histidine, a hydrated form (e.g., monohydrate including L-histidine hydrochloride monohydrate) of histidine, a salt of histidine (e.g., histidine hydrochloride) or an anhydrous form of histidine or a mixture thereof.
  • the histidine can comprise either L-histidine or D-histidine, a solvated form of histidine, a hydrated form (e.g., monohydrate including L-histidine hydrochloride monohydrate) of histidine, a salt of histidine (e.g., histidine hydrochloride) or an anhydrous form of histidine or a mixture thereof.
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein Arginine is present in a range of 1 g/L to 100 g/L, preferably 13.1 g/L to 52.2 g/L, more preferably 26.1 g/L.
  • ADM human or humanized anti-adrenomedullin
  • Arginine is present in a range of 1 g/L to 100 g/L, preferably 13.1 g/L to 52.2 g/L, more preferably 26.1 g/L.
  • the aqueous formulation comprises histidine, wherein the histidine can comprise either L-histidine or D-histidine, a solvated form of histidine, a hydrated form (e.g., monohydrate including L-histidine hydrochloride monohydrate) of histidine, a salt of histidine (e.g., histidine hydrochloride) or an anhydrous form of histidine or a mixture thereof.
  • the histidine can comprise either L-histidine or D-histidine, a solvated form of histidine, a hydrated form (e.g., monohydrate including L-histidine hydrochloride monohydrate) of histidine, a salt of histidine (e.g., histidine hydrochloride) or an anhydrous form of histidine or a mixture thereof.
  • the aqueous formulation comprises an isotonicity modifying agent which protects the antibody or protein in the formulation against freeze-thaw induced aggregation as well as aggregation on storage.
  • Said isotonicity agent can be a polyol with a molecular weight that, for example without limitation, is less than about 600 kD (e.g., in the range from about 120 to about 400 kD), and comprises multiple hydroxyl groups including sugars (e.g., reducing and nonreducing sugars or mixtures thereof, saccharide, or a carbohydrate), sugar alcohols, sugar acids, or a salt or mixtures thereof.
  • the saccharide or carbohydrate can be, for example without limitation, fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrins, soluble starch, hydroxyethyl starch, water-soluble glucans, or mixtures thereof.
  • the polyol is trehalose.
  • Subject matter of the present invention is pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein Trehalose is present in a range of 1 g/L to 100 g/L, preferably 28.4 g/L to 85.1 g/L, more preferably 56.7 g/L.
  • ADM human or humanized anti-adrenomedullin
  • Trehalose is present in a range of 1 g/L to 100 g/L, preferably 28.4 g/L to 85.1 g/L, more preferably 56.7 g/L.
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein Histidine is present in a range of 0.1 g/L to 6.4 g/L, preferably 0.8 g/L to 3.2 g/L, more preferably 1.6 g/L.
  • ADM human or humanized anti-adrenomedullin
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein the pH of the formulation is in the range of 4.0 to 8.0 preferably 5.0 to 7.0, more preferably 6.0.
  • ADM anti-adrenomedullin
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said formulation comprises an Anti-Adrenomedullin antibody directed to the N-terminal end of Adrenomedullin comprising the following sequence: SEQ ID No. 1, Arginine in a concentration of 26.1 g/L, Trehalose in a concentration of 56.7 g/L, Poloxamer in a concentration of 0.5 g/L, Histidine in a concentration of 1.6 g/L and wherein said formulation exhibits a pH of 6,0.
  • ADM human or humanized anti-adrenomedullin
  • pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said formulation comprises an Anti-Adrenomedullin antibody directed to the N-terminal end of Adrenomedullin comprising the following sequence: SEQ ID No.
  • Arginine in a concentration of 1 g/l to 100 g/L
  • Trehalose in a concentration of 1 g/l to 100 g/L
  • Poloxamer in a concentration of 0.01 g/L to 5 g/L
  • Histidine in a concentration of 0.1 g/L to 6.4 g/L and wherein said formulation exhibits a pH in a range of 4.0 to 8.0.
  • a preferred embodiment of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said formulation is stable after storage for at least 4 weeks at 2 to 8° C.
  • ADM anti-adrenomedullin
  • wt/wt-% preferably 4 wt/wt-%, more preferably 3 wt/wt-%, more preferably 2 wt/wt-%, more preferably 1 wt/wt-%, more preferably 0.05%-mol or most preferably 0.01 wt/wt-% of the total antibody is aggregated as measured for example by size exclusion high performance liquid chromatography (SEC-HPLC) and wherein said antibody is present at a total amount of 10 mg to 1000 mg, more preferably 50 mg to 700 mg and most preferably 100 mg to 500 mg referring to a 10 ml vial.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • Other methods used in the characterization of molecular weight distribution of biological macromolecules are for example Micellar liquid chromatography or ion-exchange chromatography.
  • SEC-HPLC is capable to separate protein species having apparent molecular weights of 150 kDa from others having molecular weights of 300 kDa or higher.
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein the activity of said antibody or antibody fragment in said formulation is stable after stress and wherein stress is induced by storing said formulation for at least 4 weeks at 45° C., preferably 42° C., more preferably, 38° C., more preferably 36° C. and most preferably 40° C.
  • ADM anti-adrenomedullin
  • rH relative humidity and refers to a measure of how much water vapor is in a water-air mixture compared to the maximum amount possible. Furthermore, rH is a ratio of the humidity ratio of a particular water-air mixture compared to the saturation humidity ratio at a given temperature (dry-bulb).
  • relative humidity is used according to the ICH Q1A guideline “Stability Testing of new Drug Substances and Products”. Because relative humidity is temperature dependent, precise air temperature control is required for close relative humidity control. The person skilled in the art would know that humidity is typically measured by a hygrometer such as a gravimetric hygrometer, chilled mirror hygrometer or electrolytic hygrometer.
  • “Stable” means that all critical quality attributes of the formulation remains within the specification limits of the quality specifications, in particular, the absence of any visible particulate after applied stress conditions such as storing said formulation for at least 2 weeks at 45° C., preferably 42° C., more preferably, 38° C., more preferably 36° C. and most preferably 40° C. and/or at 29° C., preferably 27° C., more preferably, 22° C., more preferably 20° C. and most preferably 25° C.
  • 70% rH preferably 65% rH, preferably 55% rH, more preferably 50% rH and most preferably 60% rH and/or at 45° C., preferably 42° C., more preferably, 38° C., more preferably 36° C. and most preferably 40° C. and/or 85% rH, preferably 80 rH, preferably 70% rH, and most preferably 75% rH or by storing said formulation at 29° C., preferably 27° C., more preferably, 22° C., more preferably 20° C. and most preferably 25° C.
  • 70% rH preferably 65% rH, preferably 55% rH, more preferably 50% rH and most preferably 60% rH or by storing said formulation at 9° C., preferably 7° C., more preferably, 2° C., more preferably 0° C. and most preferably 5° C. and/or by performing at least 5 Freeze/Thaw cycles with said formulation and/or by subjecting said formulation to mechanical stress comprising orbital shaking and overhead rotation.
  • pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein the activity of said antibody or antibody fragment in said formulation is stable after stress and wherein stress is induced by storing said formulation for at least 2 months of storage at 29° C., preferably 27° C., more preferably, 22° C., more preferably 20° C. and most preferably 25° C.
  • ADM anti-adrenomedullin
  • Another embodiment of the present invention relates to pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein the activity of said antibody or antibody fragment in said formulation is stable after stress and wherein stress is induced by storing said formulation for at least 2 weeks at 45° C., preferably 42° C., more preferably, 38° C., more preferably 36° C. and most preferably 40° C. and/or 85% rH, preferably 80 rH, preferably 70% rH, and most preferably 75% rH or by storing said formulation at 29° C., preferably 27° C., more preferably, 22° C., more preferably 20° C.
  • ADM anti-adrenomedullin
  • 1 Freeze/Thaw cycles comprises that said formulation is first frozen to ⁇ 80° C. and subsequently thawed to 25° C. at 1° C. per minute.
  • 1 to 50 preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, more preferably 2 to 8 and most preferably 5 Freeze/Thaw cycles are performed.
  • pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein the activity of said antibody or antibody fragment in said formulation is stable after stress and wherein stress is induced by subjecting said formulation to mechanical stress comprising orbital shaking and overhead rotation.
  • stress was applied by over-head rotation at 30 rpm for 24 h and/or orbital shaking at 400 rpm.
  • micron-sized protein aggregates and particles are important quality attributes of therapeutic protein formulations due to their risk of enhancing an immunogenic response. Quantification of subvisible particles larger than 10 ⁇ m and 25 m is therefore required by the pharmacopoeias and is commonly performed using light obscuration (LO) techniques.
  • LO light obscuration
  • the acceptance criteria for SVP are 6000 NMT ⁇ 10 ⁇ m/container and 600 NMT ⁇ 25 ⁇ m/container. SVPs ⁇ 10 ⁇ m have to be monitored, acceptance criteria are not defined.
  • quantification and characterization of particles with a size below 10 ⁇ m is of increasing interest and are in the meanwhile a regulatory expectation.
  • Subject matter of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said antibody is present in a concentration of 1 mg/ml to 100 mg/ml, preferably 2 mg/ml to 50 mg/ml, more preferably 10 mg/ml to 30 mg/ml, more preferably 15 mg/ml to 25 mg/ml and most preferably 20 mg/ml.
  • ADM anti-adrenomedullin
  • pharmaceutical aqueous formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said antibody or said fragment may be administered in a dose of at least 0.5 mg/Kg body weight, particularly at least 1.0 mg/kg body weight, more particularly, from 1.0 to 20.0 mg/kg body weight, e.g., from 2.0 to 10 mg/kg body weight, from 2.0 to 8.0 mg/kg body weight, or from 2.0 to 5.0 mg/kg body weight.
  • ADM anti-adrenomedullin
  • the formulation of the present invention has a shelf life of at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.
  • the aqueous formulation as disclosed by the present invention has a shelf life at 25° C. of at least or more than about 6 months, 12 months, 18 months, 24 months, 30 months, 36 months, 42 months, or 48 months (e.g. at 5° C., 25° C., or 40° C.).
  • the formulation of the present invention has a shelf life of at least about 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.
  • the formulation of the present invention has a shelf life of at least about 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.
  • physiological acceptable solution exists and that the different physiological acceptable solution can be categorized e.g., by their tonicity or purpose.
  • physiological acceptable solution's tonicity can either be isotonic, hypotonic or hypertonic.
  • the physiological acceptable solution can be selected from the group comprising Ringer's lactate, glucose solution, electrolytic solutions, isotonic sodium chloride solution.
  • Subject matter of the present invention is a ready-for-application aqueous pharmaceutical formulation obtainable by a method according to the present invention comprising said human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment in a dose of 1 to 10 mg/kg body weight.
  • ADM anti-adrenomedullin
  • Subject matter of the present invention is a ready-for-application aqueous pharmaceutical formulation obtainable by a method according to the present invention comprising said human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment in a dose of 2 to 8 mg/kg body weight, preferred 2 or 4 or 8 mg/kg body weight.
  • ADM anti-adrenomedullin
  • aqueous pharmaceutical formulation comprising an human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment wherein said antibody or said fragment is present in a concentration of at least 0.5 mg/Kg body weight, particularly at least 1.0 mg/kg body weight, more particularly, from 1.0 to 20.0 mg/kg body weight, e.g., from 2.0 to 10 mg/kg body weight, from 2.0 to 8.0 mg/kg body weight, or from 2.0 to 5.0 mg/kg body weight.
  • ADM anti-adrenomedullin
  • Subject matter of the present invention is a pharmaceutical formulation for use in therapy or prevention of an acute disease or condition selected from the group comprising: SIRS, a severe infection, sepsis, shock e.g. septic shock, acute vascular diseases as e.g. heart failure, congestion, in particular diuretic resistant congestion, inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases, cardiovascular diseases and drug-induced diseases symptoms of illness or an illness characterized by such symptoms, wherein the symptoms of illness are selected from the group of nausea, headache, muscle aches, back pain, shivering, vomiting and/or migraine.
  • an acute disease or condition selected from the group comprising: SIRS, a severe infection, sepsis, shock e.g. septic shock, acute vascular diseases as e.g. heart failure, congestion, in particular diuretic resistant congestion, inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases, cardiovascular diseases and drug-induced diseases symptoms of illness or an illness characterized by such symptoms, wherein the symptoms of illness are selected from the group of nausea, headache,
  • Acute disease or acute conditions may be selected from the group but are not limited to the group comprising severe infections as e.g. meningitis, Systemic inflammatory Response-Syndrome (SIRS), or sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as e.g. heart failure, myocardial infarction, stroke, atherosclerosis; shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, poisoning, damages induced by chemotherapy.
  • severe infections as e.g. meningitis, Systemic inflammatory Response-Syndrome (SIRS), or sepsis
  • other diseases as diabetes, cancer, acute and chronic vascular diseases as e.g. heart failure, myocardial infarction, stroke, atherosclerosis
  • shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, poisoning, damages induced by chemotherapy
  • the pharmaceutical formulation according to the present invention is for reducing the risk of mortality during sepsis and septic shock, i.e., late phases of sepsis.
  • Septic shock is a potentially fatal medical condition that occurs when sepsis, which is organ injury or damage in response to infection, leads to dangerously low blood pressure and abnormalities in cellular metabolism.
  • the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone.
  • Patients with septic shock can be clinically identified by a vasopressor requirement to maintain a mean arterial pressure of 65 mm Hg or greater and serum lactate level greater than 2 mmol/L (>18 mg/dL) in the absence of hypovolemia.
  • the primary infection is most commonly caused by bacteria, but also may be by fungi, viruses or parasites. It may be located in any part of the body, but most commonly in the lungs, brain, urinary tract, skin, or abdominal organs. It can cause multiple organ dysfunction syndrome (formerly known as multiple organ failure) and death. Frequently, people with septic shock are cared for in intensive care units. It most commonly affects children, immunocompromised individuals, and the elderly, as their immune systems cannot deal with infection as effectively as those of healthy adults. The mortality rate from septic shock is approximately 25-50%.
  • Septic shock is indicated, if there is refractory hypotension that does not respond to treatment and intravenous systemic fluid administration alone is insufficient to maintain a patient's blood pressure from becoming hypotensive also provides for an administration of a pharmaceutical formulation in accordance with the present invention.
  • Nausea When prolonged, it is a debilitating symptom. Nausea is a non-specific symptom, which means that it has many possible causes. Some common causes of nausea are motion sickness, dizziness, migraine, fainting, low blood sugar, gastroenteritis (stomach infection) or food poisoning. Nausea is a side effect of many medications including chemotherapy, or morning sickness in early pregnancy. Nausea may also be caused by anxiety, disgust and depression.
  • a migraine is a primary headache disorder characterized by recurrent headaches that are moderate to severe (for review see: Diener et al. 2012. Nat Rev Neurol. 8(3):162-71).
  • the headaches affect one half of the head, are pulsating in nature, and last from two to 72 hours.
  • Associated symptoms may include nausea, vomiting, and sensitivity to light, sound, or smell. The pain is generally made worse by physical activity. Up to one-third of people have an aura: typically, a short period of visual disturbance, which signals that the headache will soon occur. Occasionally, an aura can occur with little or no headache following it.
  • muscle aches or “muscle pain”, also termed “myalgia”, refer to a symptom of many diseases and disorders (for review see: Kyriakides et al. 2013. European Journal of Neurology 20: 997-1005).
  • Myalgia without a traumatic history is often due to Viral infections. Longer-term myalgias may be indicative of a metabolic myopathy, some nutritional deficiencies or chronic fatigue syndrome.
  • back pain refers to painful sensations in the any part of the back.
  • Episodes of back pain may be acute, sub-acute, or chronic depending on the duration.
  • the pain may be characterized as a dull ache, shooting or piercing pain, or a burning sensation.
  • the pain may radiate into the arms and hands as well as the legs or feet and may include paresthesia (tingling with no apparent cause), weakness or numbness in the legs and arms.
  • the anatomic classification of back pain follows the segments of the spine: neck pain (cervical), middle back pain (thoracic), lower back pain (lumbar) or coccydynia (tailbone or sacral pain) with the lumbar vertebrae area most common for pain.
  • the pain may originate from the muscles, nerves, bones, joints or other structures in the vertebral column (spine), however, internal structures such as the gallbladder and pancreas may also cause referred pain in the back (Cohen et al. 2008. BMJ. 33 7.′ü2 718).
  • the term “vomiting”, also known as emesis and throwing up, among other terms, is the involuntary, forcefi11 expulsion of the contents of one's stomach through the mouth and sometimes the nose (Metz A. 2017. Australian Family physician Vol. 36 (9): 688-692).
  • Vomiting can be caused by a wide variety of conditions; it may present as a specific response to ailments like gastritis or poisoning, or as a non-specific sequela of disorders ranging from brain tumors and elevated intracranial pressure to overexposure to ionizing radiation.
  • the symptoms of illnesses or diseases in a patient in need of therapy and/or prevention of such symptoms are selected from the groups of disease indications comprising inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases, cardiovascular diseases and drug-induced diseases.
  • the types of symptoms and illnesses associated herewith are provided in form of non-limiting lists. It is noted that the herein described therapy or prevention may be directed to more than one type of symptom. Further, it is noted that a given medical indication, illness, or disease may be associated with more than one of the symptoms.
  • the symptom “nausea” may be associated with illnesses inside the abdomen, e. g. obstructing disorders (for example pyloric obstruction, small bowel obstruction, colonic obstruction, superior mesenteric artery syndrome), enteric infections (for example viral or bacterial infection), inflammatory diseases (such as cholecystitis, pancreatitis, appendicitis, hepatitis), sensorimotor dysfunction (for example, gastroparesis, intestinal pseudo-obstruction, gastroesophageal reflux disease, chronic idiopathic nausea, functional vomiting, cyclic vomiting syndrome, rumination syndrome) or biliary colic; illnesses outside the abdomen, e. g.
  • obstructing disorders for example pyloric obstruction, small bowel obstruction, colonic obstruction, superior mesenteric artery syndrome
  • enteric infections for example viral or bacterial infection
  • inflammatory diseases such as cholecystitis, pancreatitis, appendicitis, hepatit
  • cardiopulmonary disorder such as cardiomyopathy, myocardial infarction
  • inner-car diseases such as motion sickness, labyrinthitis, malignancies
  • intracerebral disorders for example hemorrhage, abscess, hydrocephalus, malignancies
  • psychiatric illnesses for example anorexia and bulimia nervosa, depression
  • post-operative vomiting nausea associated with medications and drugs (for example chemotherapy and biologics therapy, antibiotics, anti-arrhythmics, digoxin, oral hypoglycemic medications, oral contraceptives), nausea associated with endocrine/metabolic diseases (such as pregnancy, uremia, ketoacidosis, thyroid and parathyroid disease, adrenal insufficiency), nausea due to intoxication because of liver failure, alcohol abuse, etc.
  • the symptom “myalgia” or “muscle pain” may be associated with injury or trauma, including sprains, hematoma, or overuse, wherein a muscle was used too much, too often, including protecting a separate injury, chronic tension, muscle pain due to rhabdomyolysis, associated with, e.g., viral infections, compression injury, drug-related, e.g., due to fibrates and statins, ACE inhibitors, cocaine, some anti-retroviral drugs, severe potassium deficiency, fibromyalgia, Ehlers-Danlos syndrome, auto-immune disorders (including for example mixed connective tissue disease, Systemic lupus erythematosus, polymyalgia rheumatic, Myositis, such as polymyositis, dermatomyositis, and inclusion body myositis, multiple sclerosis, Myalgic Encephalomyelitis (chronic fatigue syndrome), Familial Mediterranean fever, Poly
  • the symptom “headache” may be associated with primary headaches. 90% of all headaches are primary headaches. Primary headaches usually first start when people are between 20 and 40 years old. The most common types of primary headaches are migraines and tension-type headaches. They have different characteristics. Migraines typically present with pulsing head pain, nausea, photophobia (sensitivity to light) and phonophobia (sensitivity to sound). Tension-type headaches usually present with non-pulsing “bandlike” pressure on both sides of the head, not accompanied by other symptoms. Other very rare types of primary headaches include cluster headaches: short episodes (15-180 minutes) of severe pain, usually around one eye, with autonomic symptoms (tearing, red eye, nasal congestion) which occur at the same time every day.
  • Cluster headaches can be treated with triptans and prevented with prednisone, ergotamine or lithium; trigeminal neuralgia or occipital neuralgia characterized by shooting face pain, hemicrania continua, i.e., continuous unilateral pain with episodes of severe pain; primary stabbing headaches, e.g., recurrent episodes of stabbing “ice pick pain” or “jabs and jolts” for 1 second to several minutes without autonomic symptoms (tearing, red eye, nasal congestion); Primary cough headache that starts suddenly and lasts for several minutes after coughing, sneezing or straining (anything that may increase pressure in the head).
  • trigeminal neuralgia or occipital neuralgia characterized by shooting face pain, hemicrania continua, i.e., continuous unilateral pain with episodes of severe pain
  • primary stabbing headaches e.g., recurrent episodes of stabbing “ice pick pain” or “jabs and jolts” for 1 second to several minutes
  • headache is defined as primary or secondary headache.
  • Primary headache is defined as migraine, tension-type headache (TTH), cluster headache and other trigeminal autonomic cephalalgias, and other primary headaches.
  • TTH tension-type headache
  • cluster headache and other trigeminal autonomic cephalalgias
  • Other primary headaches Diagnosis or assessment of headache is well-established in the art. Assessment may be performed based on subjective measures, such as patient characterization of symptoms.
  • the symptom “vomiting” may be associated with gastritis (inflammation of the gastric wall), gastroenteritis, gastroesophageal reflux disease, celiac disease, non-celiac gluten sensitivity, pyloric stenosis, bowel obstruction, overeating, acute abdomen and/or peritonitis, ileus, food allergies (often in conjunction with hives or swelling); cholecystitis, pancreatitis, appendicitis, hepatitis; food poisoning, allergic reaction to cow's milk proteins, e.g., milk allergy or lactose intolerance; motion sickness, Mérier's disease, concussion, cerebral hemorrhage, migraine, brain tumors, benign intracranial hypertension and hydrocephalus, metabolic disturbances such as hypercalcemia, Uremia adrenal insufficiency, hypoglycemia, hyperglycemia, drug reaction, alcohol intoxication, opioid uptake, selective serotonin reuptake inhibitors; use
  • Another embodiment of the invention relates to an anti-ADM antibody or an anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of symptoms of illness selected from the group of nausea, headache, muscle aches, back pain, shivering, vomiting or for the use in therapy or prevention of illnesses characterized by such symptoms, such as migraine, in a subject in need thereof according to any of the preceding embodiments to be used in combination with known medicaments against nausea.
  • Another embodiment of the invention relates to an anti-ADM antibody or an anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of symptoms of illness selected from the group of nausea, headache, muscle aches, back pain, shivering, vomiting or for the use in therapy or prevention of illnesses characterized by such symptoms, such as migraine, in a subject in need thereof according to any of the preceding embodiments to be used in combination with known medicaments against myalgia.
  • Another embodiment of the invention relates to an anti-ADM antibody or an anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of symptoms of illness selected from the group of nausea, headache, muscle aches, back pain, shivering, vomiting or for the use in therapy or prevention of illnesses characterized by such symptoms, such as migraine, in a subject in need thereof according to any of the preceding embodiments to be used in combination with known medicaments against back pain.
  • Organic dysfunction denotes a condition or a state of health where an organ does not perform its expected function.
  • Organic failure denotes an organ dysfunction to such a degree that normal homeostasis cannot be maintained without external clinical intervention
  • the patient group(s) addressed by the instant invention can be defined as set out below.
  • the criteria orientate on the clinical SOFA score.
  • the SOFA is a six-organ dysfunction/failure score measuring multiple organ failure daily. Each organ is graded from 0 (normal) to 4 (the most abnormal), providing a daily score of 0 to 24 points.
  • the objective of the SOFA is to create a simple, reliable, and continuous score for clinical staff.
  • MAP mean arterial pressure
  • CNS central nervous system
  • SaO 2 peripheral arterial oxygen saturation
  • a PaO 2 /FIO 2 ratio was used preferentially. If not available, the SaO 2 /FIO 2 ratio was used; b vasoactive mediations administered for at least 1 hr (dopamine and norepinephrine ⁇ g/kg/min).
  • the patient group pursuant to the invention is having as lower threshold at least one SOFA score, being it 1 for one the clinical criteria respiration, or liver, or coagulation, or cardiovascular, or CNS, or renal on day of admission to hospital or Intensive Care Unit (ICU).
  • said patient group is in need of therapeutic intervention pursuant to the invention, and thus in need for prevention or reduction of organ dysfunction or organ failure
  • the patient group pursuant to the invention is having as lower threshold at least two SOFA scores, being it 1 each for the clinical criteria respiration, and/or liver, and/or coagulation, and/or cardiovascular, and/or CNS, and/or renal on day of admission to hospital or Intensive Care Unit (ICU).
  • said patient group is in need of therapeutic intervention pursuant to the invention, and thus in need for prevention or reduction of organ dysfunction or organ failure.
  • the patient group pursuant to the invention is having as lower threshold at least three SOFA scores, being it 1 each for the clinical criteria respiration, and/or liver, and/or coagulation, and/or cardiovascular, and/or CNS, and/or renal on day of admission to hospital or Intensive Care Unit (ICU).
  • said patient group is in need of therapeutic intervention pursuant to the invention, and thus in need for prevention or reduction of organ dysfunction or organ failure.
  • the patient group pursuant to the invention is having as lower threshold at least four SOFA scores, being it 1 each for the clinical criteria respiration, and/or liver, and/or coagulation, and/or cardiovascular, and/or CNS, and/or renal on day of admission to hospital or Intensive Care Unit (ICU).
  • said patient group is in need of therapeutic intervention pursuant to the invention, and thus in need for prevention or reduction of organ dysfunction or organ failure.
  • said clinical criteria denote the patient group(s) for kidney dysfunction/failure:
  • the patient group for liver dysfunction/failure is characterized by a lower threshold of Bilirubin of >1.2 mg/dL, preferably >1.9 mg/dL, more preferably >5.9 mg/dL.
  • the pharmaceutical formulation may be also administered preventively before the patient exhibits any signs of dysfunction or failure of an organ. This might be the case if the patient has a chronic or acute disease or acute condition where dysfunction or failure problems may be expected, e.g. comprising severe infections as e.g. meningitis, Systemic inflammatory Response-Syndrome (SIRS,) sepsis; other diseases as diabetes, cancer, acute and chronic vascular diseases as e.g. heart failure, myocardial infarction, stroke, atherosclerosis; shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunction, burnings, surgery, traumata, poisoning.
  • the pharmaceutical formulation may be also administered preventively or therapeutically before, or during or after chemotherapy.
  • ischemic damages may occur to certain organs which may result in dysfunction or failure of an organ.
  • Preventively means before an organ damage occurs and therapeutically means that an organ damage has been already occurred.
  • the antibody or fragment or scaffold according to the present invention for reducing the risk of organ dysfunction or failure during sepsis and septic shock, i.e., late phases of sepsis.
  • Systemic circulation refers to the part of the circulatory system in which the blood leaves the heart, services the body's cells, and then re-enters the heart.
  • Blood leaves through the left ventricle to the aorta, the body's largest artery.
  • the aorta leads to smaller arteries, arterioles, and finally capillaries. Waste and carbon dioxide diffuse out of the cell into the blood, and oxygen in the blood diffuses into the cell. Blood then moves to venous capillaries, and then to the venae cavae: the lower inferior vena cava and the upper superior vena cava, through which the blood re-enters the heart at the right atrium.
  • stabilizing the circulation means stabilizing the systemic circulation.
  • the term systemic circulation would not encompass phenomena of microcirculation.
  • Microcirculation is the delivery of fresh blood to the smallest blood vessels, present in the vasculature embedded within organ tissues. This contrasts with macrocirculation, which transport blood to and from the organs.
  • the state of the systemic circulation may be measures by parameters like mean arterial pressure, blood pressure (other parameters see above).
  • a patient in need for stabilizing the circulation may be, thus a patient that exhibits a heart rate of >100 beats/min and or ⁇ 65 mm Hg mean arterial pressure.
  • an anti-Adrenomedullin (ADM) antibody or by an anti-ADM antibody fragment binding to adrenomedullin or an anti-ADM non-Ig scaffold binding to adrenomedullin, this can be measured and is characterized by an increase of the mean arterial pressure over 65 mm Hg and/or a decrease of heart rate under 100 beats/min.
  • ADM anti-Adrenomedullin
  • the pharmaceutical formulation according to the present invention reduces the vasopressor-agents requirement, e.g., catecholamine requirement, of said patient.
  • the vasopressor-agents requirement, e.g., catecholamine requirement of a patient is an indicator for the condition of the circulation of said patient.
  • the pharmaceutical formulation may be administered at a point of time when the patient is in need of a vasopressor agent, e.g., catecholamine.
  • said patient is a patient in need of increasing the blood pressure.
  • ADM in plasma binds to endothelial cells and thereby stabilizes or even restores vascular integrity.
  • this function is strengthened, when plasma ADM levels increase as a consequence of administration of the antibody, which is a non-neutralizing antibody.
  • binding of the antibody to ADM reduces the proteolytic decay of ADM.
  • said pharmaceutical formulation is for use in intervention and therapy of congestion in a patient according to any embodiment of the invention, wherein said patient is resistant against diuretics or is a non-responder to diuretics therapy.
  • Another specific embodiment of the invention relates to said pharmaceutical formulation for use in intervention and therapy of congestion in a patient in need thereof and wherein said patient is resistant against diuretics or is a non-responder to diuretics therapy.
  • diuretic resistance is defined in general as failure to decrease the extracellular fluid volume despite liberal use of diuretics (Ravnan et al. 2002. CHF 8:80-85).
  • Epstein et al. defined diuretic resistance as a failure to excrete at least 90 mmol of sodium within 72 hours of a 160-mg oral furosemide dose given twice daily (Epstein et al. 1977. Curr Ther Res. 21:656-667).
  • Adaptation to diuretic drugs and diuretic resistance may be caused by similar mechanisms.
  • Diuretic adaptations can be classified as those that occur during diuretic action, those that cause sodium retention in the short term (causing ‘post-diuretic NaCl retention’), and those that increase sodium retention chronically (the ‘braking phenomenon’).
  • Ways in which kidneys adapt to chronic diuretic treatment are: First, nephron segments downstream from the site of diuretic action increase NaCl reabsorption during diuretic administration because delivered NaCl load is increased. Second, when diuretic concentrations in the tubule decline, the kidney tubules act to retain Na until the next dose of diuretic is administered.
  • diuretic resistance is thought to occur in one of three patients with congestive HF.
  • Heart failure represents the most common clinical situation in which diuretic resistance is observed.
  • diuretic resistance is not commonly encountered, as long as renal function is preserved.
  • diuretic resistance occurs more frequently and often becomes a clinical problem (Brater 1985. Drugs 30:427-443; Taylor 2000 Cardiol Rev. 8:104-114).
  • Subject matter of the present invention is a pharmaceutical formulation for use in therapy or prevention comprising:
  • fragments of pre-pro-Adrenomedullin that may be determined in a bodily fluid is/are selected from the group comprising:
  • the level of the fragments of pre-proADM and/or fragments thereof is determined by using at least one binder, wherein said binder binds to a region comprised within the sequence of MR-proADM (SEQ ID No. 11).
  • the level of the fragments of pre-proADM and/or fragments thereof is determined by using at least one binder, wherein said binder binds to a region comprised within the sequence of PAMP (SEQ ID No. 10).
  • the level of mature ADM-NH 2 (SEQ ID No. 13) and/or ADM 1-52-Gly (SEQ ID No. 14)—immunoreactivity or the level of MR-proADM (SEQ ID No. 11) immunoreactivity or the level of CT-proADM (SEQ ID No. 12) immunoreactivity is determined and correlated with the need of said patient for therapy or intervention, wherein said patient is identified as having such a need if the level of mature ADM-NH 2 (SEQ ID No. 13) and/or ADM 1-52-Gly (SEQ ID No. 14)—immunoreactivity or the level of MR-proADM (SEQ ID No. 11) immunoreactivity or the level of CT-proADM (SEQ ID No. 12) immunoreactivity in the bodily fluid of said subject is above a threshold.
  • the level of proADM and/or fragments thereof is determined by using at least one binder selected from the group: a binder that binds to a region comprised within the following sequence of mature ADM-NH 2 (SEQ ID No. 13) and/or ADM1-52-Gly (SEQ ID No. 14) and a second binder that binds to a region comprised within the sequence of mature ADM-NH 2 (SEQ ID NO. 13) and/or ADM 1-52-Gly (SEQ ID No. 14).
  • the level of proADM and/or fragments thereof is determined by using at least one binder selected from the group: a binder that binds to a region comprised within the sequence of MR-proADM (SEQ ID No. 11) and a second binder that binds to a region comprised within the sequence of MR-proADM (SEQ ID No. 11).
  • the level of pro-ADM and/or fragments thereof is determined by using at least one binder selected from the group: a binder that binds to a region comprised within the sequence of CT-proADM (SEQ ID No. 12) and a second binder that binds to a region comprised within the sequence of CT-pro-ADM (SEQ ID No. 12).
  • Subject matter in a particular embodiment of the present diagnostic method is a method, according to the present invention wherein said fragment may be selected from MR-proADM according to SEQ ID No.: 11 or mature ADM-NH 2 according to SEQ ID No.: 13.
  • the predetermined threshold of Bio-ADM in a sample of bodily fluid of said subject is between 40 and 100 ⁇ g/mL, more preferred between 50 and 90 ⁇ g/mL, even more preferred between 60 and 80 ⁇ g/mL, most preferred said threshold is 70 ⁇ g/mL.
  • the threshold is within a threshold range for plasma CT-proADM that is between 85 and 350 ⁇ mol/L, preferably between 100 and 250 ⁇ mol/L, most preferred a threshold of 150 ⁇ mol/L is applied.
  • the threshold is within a threshold range for plasma ADM-NH 2 that is between 50 and 100 ⁇ g/ml, preferably between 60 and 90 ⁇ g/ml, most preferred a threshold of 70 ⁇ g/ml is applied.
  • a threshold for plasma CT-proADM is the 5fold median concentration, preferably the 4fold median concentration, more preferred the 3fold median concentration, most preferred the 2fold median concentration of a normal healthy population.
  • a threshold for plasma ADM-Gly is the 5fold median concentration, preferably the 4fold median concentration, more preferred the 3fold median concentration, most preferred the 2fold median concentration of a normal healthy population.
  • a threshold for plasma PAMP is the 5fold median concentration, preferably the 4fold median concentration, more preferred the 3fold median concentration, most preferred the 2fold median concentration of a normal healthy population.
  • Subject matter of the present diagnostic method is a method according to the diagnostic method invention, wherein the level of Pro-Adrenomedullin or fragments thereof of at least 5 amino acids is determined by using a binder to Pro-Adrenomedullin or fragments thereof of at least 5 amino acids.
  • Subject matter of the present invention is a pharmaceutical formulation for use in therapy or prevention for use in therapy of a patient with shock, in particular septic shock, wherein said patient:
  • the term “admission to ICU” refers to patients admitted to intensive care are patients who have, or are likely to have, one or several acute, directly life-threatening malfunctions which require the use of organ supporting methods. Criteria for admitting patients to intensive care units have been developed which are well documented in the art (Nates et al. (2016), Critical care medicine 44: 1553-1602). The term “admission to ICU” shall encompass admissions under these criteria.
  • adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is for use in therapy of a patient with shock, in particular septic shock, wherein said patient:
  • SEQ ID NO. 1 YRQSMNNFQGLRSFGCRFGTC wherein, when the patient has suffered from shock, in particular from a septic shock a) not longer than 10 hours and has been admitted to ICU b) not longer than 10 hours, the starting point of treatment with said Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is the shortest of a) and b).
  • ADM Anti-adrenomedullin
  • the starting point of treatment with the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is the shortest of b) and c).
  • the starting point of treatment with the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is the shortest of a), b) and c).
  • the patient has suffered from shock, in particular from a septic shock, not longer than 8.4, preferably 8.26 (0.344 days) at the starting point of treatment with said Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.
  • shock in particular from a septic shock, not longer than 8.4, preferably 8.26 (0.344 days) at the starting point of treatment with said Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.
  • ADM Anti-adrenomedullin
  • the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold can be administered a) within 10 hours after occurrence of shock in said patient and c) before the patient has received organ support or not longer than 10h of organ support, the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered at the shortest of a) and c).
  • the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold can be administered b) within 10 hours after admission of said patient to ICU and c) before the patient has received organ support or not longer than 10h of organ support, the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered at the shortest of b) and c).
  • the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 9, preferably 8.4, preferably 8.26 (0.344 days), preferably 8, preferably 7, preferably 6, preferably 5.76 (0.25 days), preferably 5.75 (0.25 days), preferably 5, preferably 4, preferably 3 hours after occurrence of shock and/or sepsis in said patient.
  • ADM Anti-adrenomedullin
  • the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 8.4, preferably 8.26 (0.344 days) hours after occurrence of shock and/or sepsis in said patient.
  • the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 9, preferably 8.4, preferably 8.26 (0.344 days), preferably 8, preferably 7, preferably 6, preferably 5.76 (0.25 days), preferably 5.75 (0.25 days), preferably 5, preferably 4, preferably 3 hours after admission of said patient to ICU.
  • ADM Anti-adrenomedullin
  • the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 9, preferably 8.4, preferably 8.26 (0.344 days), preferably 8, preferably 7, preferably 6, preferably 5.76 (0.25 days), preferably 5.75 (0.25 days), preferably 5, preferably 4, preferably 3 hours after the patient has received organ support at the starting point of treatment with said Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.
  • the Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered 8.4, preferably 8.26 (0.344 days) hours after the patient has received organ support at the starting point of treatment with said Anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.
  • the shock is a septic shock, shock due to Covid-19, shock due to burns or a traumatic shock.
  • Subject matter of the present invention is a pharmaceutical formulation for use in therapy or prevention for use in therapy wherein said patient has shock that is selected from the group comprising shock due to hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock, septic shock, shock due to Covid-19, shock due to burns and traumatic shock.
  • a further embodiment of the invention is a pharmaceutical formulation for use in therapy or prevention for use in therapy wherein said patient has shock due chemical contamination with acidic, basic or oxidative reagents and chemicals in solid, liquid or gaseous form.
  • shock is characterized by decreased oxygen delivery and/or increased oxygen consumption or inadequate oxygen utilization leading to cellular and tissue hypoxia. It is a life-threatening condition of circulatory failure and most commonly manifested as hypotension (systolic blood pressure less than 90 mm Hg or MAP less than 65 mmHg). Shock is divided into four main types based on the underlying cause: hypovolemic, cardiogenic, obstructive, and distributive shock (Vincent and De Backer 2014. N. Engl. J. Med. 370(6): 583).
  • Established criteria for the diagnosis of CS are: (i) systolic blood pressure, ⁇ 90 mmHg for >30 min or vasopressors required to achieve a blood pressure ⁇ 90 mmHg; (ii) pulmonary congestion or elevated left-ventricular filling pressures; (iii) signs of impaired organ perfusion with at least one of the following criteria: (a) altered mental status; (b) cold, clammy skin; (c) oliguria ( ⁇ 0.5 mL/kg/h or ⁇ 30 mL/h); (d) increased serum-lactate (Reynolds and Hochman 2008. Circulation 117: 686-697).
  • AMI Acute myocardial infarction
  • Non-AMI-related CS may be caused by decompensated valvular heart disease, acute myocarditis, arrhythmias, etc. with heterogeneous treatment options. This translates in 40 000 to 50 000 patients per year in the USA and 60 000 to 70 000 in Europe.
  • hypovolemic shock refers to a shock where the patient may have suffered a hemorrhagic disease including gastrointestinal bleed, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor eroding into a major blood vessel) and spontaneous bleeding in the setting of anticoagulant use or a non-hemorrhagic disease including vomiting, diarrhea, renal loss, skin losses/insensible losses (e.g. burns, heat stroke) or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma.
  • a hemorrhagic disease including gastrointestinal bleed, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor eroding into a major blood vessel) and spontaneous bleeding in the setting of anticoagulant use or a non-hemorrhagic disease including vomiting, diarrhea, renal loss, skin losses/insensible losses (e.g. burns,
  • obstructive shock refers to a shock where the patient may have suffered a cardiac tamponade, tension pneumothorax, pulmonary embolism or aortic stenosis.
  • Obstructive shock is due to a physical obstruction of the great vessels or the heart itself.
  • Several conditions can result in this form of shock (e.g., cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis).
  • cardiac tamponade tension pneumothorax
  • pulmonary embolism aortic stenosis
  • TSS toxic shock syndrome
  • anaphylaxis a sudden, severe allergic reaction
  • adrenal insufficiency acute worsening of chronic adrenal insufficiency, destruction or removal of the adrenal glands, suppression of adrenal gland function due to exogenous steroids, hypopituitarism and metabolic failure of hormone production
  • reactions to drugs or toxins heavy metal poisoning
  • hepatic liver insufficiency and damage to the central nervous system.
  • Refractory shock has been defined as requirement of noradrenaline infusion of >0.5 ⁇ g/kg/min despite adequate volume resuscitation. Mortality in these patients may be as high as 94% and the assessment and management of these patients requires a much more aggressive approach for survival.
  • the term “refractory shock” is used when the tissue perfusion cannot be restored with the initial corrective measures employed (e.g., vasopressors) and may therefore be referred to as “high vasopressor-dependent” or “vasopressor-resistant” shock (Udupa and Shetty 2018. Indian J Respir Care 7: 67-72).
  • Patients with refractory shock may have features of inadequate perfusion such as hypotension (mean arterial blood pressure ⁇ 65 mmHg), tachycardia, cold peripheries, prolonged capillary refill time, and tachypnea consequent to the hypoxia and acidosis. Fever may be seen in septic shock. Other signs of hypoperfusion such as altered sensorium, hyperlactatemia, and oliguria may also be seen. These well-known signs of shock are not helpful in identifying whether the problem is at the pump (heart) or circuitry (vessels and tissues). Different types of shock can coexist, and all forms of shock can become refractory, as evidenced by unresponsiveness to high-dose vasopressors (Udupa and Shetty 2018. Indian J Respir Care 7: 67-72).
  • Subject matter of the present invention is a pharmaceutical formulation for use in therapy or prevention wherein the level of DPP3 is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to DPP3.
  • One embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein said shock is selected from the group comprising shock due to hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.
  • Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein
  • One specific embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein the level of DPP3 is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to DPP3.
  • Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein either the level of DPP3 protein and/or the level of active DPP3 is determined and compared to a predetermined threshold.
  • One embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein said patient is additionally characterized by having a level of ADM-NH 2 above a threshold.
  • the level of ADM —NH2 is measured in order to identify patients in shock.
  • One preferred embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein said threshold of ADM-NH 2 in a sample of bodily fluid of said patient is between 40 and 100 ⁇ g/mL, more preferred between 50 and 90 ⁇ g/mL, even more preferred between 60 and 80 ⁇ g/mL, most preferred said threshold is 70 ⁇ g/mL.
  • Another embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein the level of ADM-NH 2 is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to ADM-NH 2 .
  • Another preferred embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in therapy or prevention of shock in a patient, wherein the sample of bodily fluid of said patient is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.
  • the sample of bodily fluid of said patient is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.
  • either the level of DPP3 protein and/or the level of active DPP3 is determined and compared to a threshold level.
  • the level of DPP3 as the amount of DPP3 protein and/or DPP3 activity in a sample of bodily fluid of said subject may be determined by different methods, e.g., immunoassays, activity assays, mass spectrometric methods etc.
  • DPP3 activity can be measured by detection of cleavage products of DPP3 specific substrates.
  • peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, valorphin, ⁇ -casomorphin, dynorphin, proctolin, ACTH (Adrenocorticotropic hormone) and MSH (melanocyte-stimulating hormone; Abrami ⁇ et al. 2000, Bar ⁇ un et al. 2007, Dhanda et al. 2008).
  • the cleavage of mentioned peptide hormones as well as other untagged oligopeptides e.g., Ala-Ala-Ala-Ala, Dhanda et al.
  • Detection methods include, but are not limited to, HPLC analysis (e.g., Lee & Snyder 1982), mass spectrometry (e.g., Abrami ⁇ et al. 2000), H1-NMR analysis (e.g., Vandenberg et al. 1985), capillary zone electrophoresis (CE; e.g. Bar ⁇ un et al. 2007), thin layer chromatography (e.g. Dhanda et al. 2008) or reversed phase chromatography (e.g. Mazocco et al. 2006).
  • HPLC analysis e.g., Lee & Snyder 1982
  • mass spectrometry e.g., Abrami ⁇ et al. 2000
  • H1-NMR analysis e.g., Vandenberg et al. 1985
  • CE capillary zone electrophoresis
  • thin layer chromatography e.g. Dhanda et al. 2008
  • reversed phase chromatography e.g. Mazocco et al
  • DPP3 carrying samples can be immobilized and divided on a gel by electrophoresis, gels stained with fluorogenic substrate (e.g., Arg-Arg- ⁇ NA) and Fast Garnet GBC and fluorescent protein bands detected by a fluorescence reader (Ohkubo et al. 1999).
  • fluorogenic substrate e.g., Arg-Arg- ⁇ NA
  • DPP3 activity is a Protease-Glom Assay (commercially available at Promega).
  • DPP3 specific di- or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) are coupled to aminoluciferin.
  • aminoluciferin Upon cleavage by DPP3, aminoluciferin is released and serves as a substrate for a coupled luciferase reaction that emits detectable luminescence.
  • solid phase may be used to include any material or vessel in which or on which the assay may be performed and includes, but is not limited to: porous materials, nonporous materials, test tubes, wells, slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particals (e.g. Dynabeadsm or Piercerm magnetic beads from Thermo Fisher Scientific), etc.
  • agarose resins e.g., Sepharose from GE Healthcare Life Sciences
  • magnetic particals e.g. Dynabeadsm or Piercerm magnetic beads from Thermo Fisher Scientific
  • the amount of DPP3 protein and/or DPP3 activity in a sample of bodily fluid of said subject may be determined for example by one of the following methods:
  • the ECA is a DPP3-specific activity assay that uses black high-binding polystyrene microtiter plates as solid phase. These plates are coated with monoclonal anti-DPP3 antibody AK2555 (capture antibody). Twenty microliters of samples (e.g., serum, heparin-plasma, citrate-plasma, EDTA-plasma, cerebrospinal fluid and urine) and calibrators are pipetted into coated black microtiter plates. After adding assay buffer (200 ⁇ L), the microtiter plates are incubated for 2 h at 22° C. and 600 rpm. DPP3 present in the samples is immobilized by binding to the capture antibody. Unbound sample components are removed by 4 washing steps (350 ⁇ L per well).
  • samples e.g., serum, heparin-plasma, citrate-plasma, EDTA-plasma, cerebrospinal fluid and urine
  • calibrators are pipetted into coated black microtiter plates. After adding assay buffer
  • the specific activity of immobilized DPP3 is measured by the addition of the fluorogenic substrate, Arg-Arg- ⁇ -Naphthylamide (Arg2- ⁇ NA), in reaction buffer followed by incubation at 37° C. for 1 h. DPP3 specifically cleaves Arg2- ⁇ NA into Arg-Arg dipeptide and fluorescent ⁇ -naphthylamine. Fluorescence is measured with a fluorometer using an excitation wavelength of 340 nm and emission is detected at 410 nm. The activity of DPP3 is determined with a 6-point calibration curve. Calibrators and samples are preferably run in duplicates.
  • samples e.g., serum, heparin-plasma, citrate-plasma
  • calibrators are pipetted into
  • an assay is used for determining the level of DPP3, wherein the assay sensitivity of said assay is able to quantify the DPP3 of healthy subjects and is ⁇ 20 ng/ml, preferably ⁇ 30 ng/ml and more preferably ⁇ 40 ng/ml.
  • the IB10 Sphingotest® DPP3 is a rapid point-of-care (POC) immunoassay for the in vitro quantitative determination of Dipeptidyl Peptidase 3 (DPP3) in human EDTA whole blood and plasma.
  • POC point-of-care
  • the Nexus IB10 immunochemistry system combines chemistry with microfluidics and centrifugal flow to rapidly prepare a cell free plasma from whole blood that can then be moved through a channel to rehydrate, solubilize, and mix with freeze dried immunoconjugates
  • said sample of bodily fluid is selected from the group of whole blood, plasma, and serum.
  • a bodily fluid according to the present invention is in one particular embodiment a blood sample.
  • a blood sample may be selected from the group comprising whole blood, serum and plasma.
  • said sample is selected from the group comprising human citrate plasma, heparin plasma and EDTA plasma.
  • an assay is used for determining the level ADM-NH 2 , wherein the assay sensitivity of said assay is able to quantify the mature ADM-NH 2 of healthy subjects and is ⁇ 70 ⁇ g/ml, preferably ⁇ 40 ⁇ g/ml and more preferably ⁇ 10 ⁇ g/ml.
  • said binder exhibits a binding affinity to ADM-NH 2 of at least 10 7 M ⁇ 1 , preferred 10 8 M ⁇ 1 , preferred affinity is greater than 10 9 M ⁇ 1 , most preferred greater than 10 10 M ⁇ 1 .
  • a person skilled in the art knows that it may be considered to compensate lower affinity by applying a higher dose of compounds and this measure would not lead out-of-the-scope of the invention.
  • the kinetics of binding of Adrenomedullin to immobilized antibody was determined by means of label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using an anti-mouse Fc antibody covalently coupled in high density to a CM5 sensor surface according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare), (Lorenz et al. 2011. Antimicrob Agents Chemother. 55 (1): 165-173).
  • binding affinity refers to the affinity of the proteins described in the present invention to their binding targets and is expressed numerically using “Kd” values.
  • Kd is intended to refer to the dissociation constant of an antibody-antigen interaction. If two or more proteins are indicated to have comparable binding affinities towards their binding targets, then the Kd values for binding of the respective proteins towards their binding targets, are within ⁇ 2-fold of each other. If two or more proteins are indicated to have comparable binding affinities towards single binding target, then the Kd values for binding of the respective proteins towards said single binding target, are within ⁇ 2-fold of each other.
  • said binder is selected from the group comprising an antibody or an antibody fragment or a non-Ig scaffold binding to ADM-NH 2 .
  • such assay for determining the level of the biomarkers is a sandwich immunoassay using any kind of detection technology including but not restricted to enzyme label, chemiluminescence label, electrochemiluminescence label, preferably a fully automated assay.
  • a sandwich immunoassay using any kind of detection technology including but not restricted to enzyme label, chemiluminescence label, electrochemiluminescence label, preferably a fully automated assay.
  • an assay is an enzyme labeled sandwich assay.
  • Examples of automated or fully automated assay comprise assays that may be used for one of the following systems: Roche Elecsys®, Abbott Architect@, Siemens Centauer®, Brahms Kryptor®, BiomerieuxVidas®, Alere Triage®.
  • immunoassays are known and may be used for the assays and methods of the present invention, these include: radioimmunoassays (“RIA”), homogeneous enzyme-multiplied immunoassays (“EMIT”), enzyme linked immunoadsorbent assays (“ELISA”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays and immuno-chromatography assays.
  • RIA radioimmunoassays
  • EMIT homogeneous enzyme-multiplied immunoassays
  • ELISA enzyme linked immunoadsorbent assays
  • ARIS apoenzyme reactivation immunoassay
  • dipstick immunoassays dipstick immunoassays and immuno-chromatography assays.
  • such an assay is a sandwich immunoassay using any kind of detection technology including but not restricted to enzyme label, chemiluminescence label, electrochemiluminescence label, preferably a fully automated assay.
  • such an assay is an enzyme labeled sandwich assay. Examples of automated or fully automated assay comprise assays that may be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.
  • it may be a so-called POC-test (point-of-care) that is a test technology, which allows performing the test within less than 1 hour near the patient without the requirement of a fully automated assay system.
  • POC-test point-of-care
  • One example for this technology is the immunochromatographic test technology.
  • the assays can be homogenous or heterogeneous assays, competitive and non-competitive assays.
  • the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, wherein the molecule to be detected and/or quantified is bound to a first antibody and to a second antibody.
  • the first antibody may be bound to a solid phase, e.g., a bead, a surface of a well or other container, a chip or a strip
  • the second antibody is an antibody which is labeled, e.g., with a dye, with a radioisotope, or a reactive or catalytically active moiety.
  • the amount of labeled antibody bound to the analyte is then measured by an appropriate method.
  • the general composition and procedures involved with “sandwich assays” are well-established and known to the skilled person ( The Immunoassay Handbook , Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 February; 10(1):4-10. PMID: 16376134).
  • the assay comprises two capture molecules, preferably antibodies which are both present as dispersions in a liquid reaction mixture, wherein a first labelling component is attached to the first capture molecule, wherein said first labelling component is part of a labelling system based on fluorescence- or chemiluminescence-quenching or amplification, and a second labelling component of said marking system is attached to the second capture molecule, so that upon binding of both capture molecules to the analyte a measurable signal is generated that allows for the detection of the formed sandwich complexes in the solution comprising the sample.
  • said labeling system comprises rare earth cryptates or rare earth chelates in combination with fluorescence dye or chemiluminescence dye, in particular a dye of the cyanine type.
  • fluorescence based assays comprise the use of dyes, which may for instance be selected from the group comprising FAM (5- or 6-carboxyfluorescein), VIC, NED, Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700/800, Cyanine dyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen, 6-Carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET, 6-Carboxy-4′,5′-dichloro-2′,7′-dimethodyfluorescein (JOE), N,N,N′,N′-Tetramethyl-6-carboxyrhodamine (TAMRA), 6-Carboxy
  • chemiluminescence based assays comprise the use of dyes, based on the physical principles described for chemiluminescent materials in (Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, J. I Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, p. 518-562, incorporated herein by reference, including citations on pages 551-562).
  • Preferred chemiluminescent dyes are acridiniumesters.
  • an “assay” or “diagnostic assay” can be of any type applied in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to one or more capture probes with a certain affinity. Concerning the interaction between capture molecules and target molecules or molecules of interest, the affinity constant is preferably greater than 10 8 M ⁇ 1 .
  • One possibility of quantifying the difference in calibration is a method comparison analysis (correlation) of the assay in question with the respective biomarker assay used in the present invention by measuring the respective biomarker (e.g., bio-ADM, DPP3) in samples using both methods.
  • Another possibility is to determine with the assay in question, given this test has sufficient analytical sensitivity, the median biomarker level of a representative normal population, compare results with the median biomarker levels as described in the literature and recalculate the calibration based on the difference obtained by this comparison.
  • the level of ADM —NH2 is therefore measured in order to identify patients having an increased risk of running into shock.
  • Adrecizumab an N-terminal anti-ADM antibody
  • Adrecizumab a humanized version of an N-terminal anti-ADM antibody
  • beneficial effects of Adrecizumab on vascular barrier function and survival were recently demonstrated in preclinical models of systemic inflammation and sepsis (Geven et al. 2018. Shock 50(6):648-654).
  • pre-treatment with Adrecizumab attenuated renal vascular leakage in endotoxemic rats as well as in mice with CLP-induced sepsis, which coincided with increased renal expression of the protective peptide Ang-1 and reduced expression of the detrimental peptide vascular endothelial growth factor.
  • Adrecizumab pre-treatment with Adrecizumab improved 7-day survival in CLP-induced sepsis in mice from 10 to 50% for single and from 0 to 40% for repeated dose administration. Moreover, in a phase I study, excellent safety and tolerability was demonstrated (see Example 6): no serious adverse events were observed, no signal of adverse events occurring more frequently in Adrecizumab-treated subjects was detected and no relevant changes in other safety parameters were found (Geven et al. 2017. Intensive Care Med Exp 5 (Suppl 2): 0427). Of particular interest is the proposed mechanism of action of Adrecizumab. Both animal and human data reveal a potent, dose-dependent increase of circulating ADM following administration of this antibody. Based on pharmacokinetic data and the lack of an increase in MR-proADM (an inactive peptide fragment derived from the same prohormone as ADM), the higher circulating ADM levels cannot be explained by an increased production.
  • MR-proADM an inactive peptide fragment derived from the same prohormone
  • a mechanistic explanation for this increase could be that the excess of antibody in the circulation may drain ADM from the interstitium to the circulation, since ADM is small enough to cross the endothelial barrier, whereas the antibody is not (Geven et al. 2018. Shock. 50(2):132-140 and Voors et al (J. Eur J Heart Fail. 2019 February; 21(2):163-171)). In addition, binding of the antibody to ADM leads to a prolongation of ADM's half-life.
  • Adrecizumab specifically. There is no reason to doubt that what is true for Adrecizumab will also be true for antibodies sharing main essential features (in particular affinity and epitope specificity) Antibodies that target the same region must be expected to have the same technical effect, provided they have the same affinity and same or very comparable structural features (size, shape, . . . ).
  • antibody generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain-antibodies” (Bird et al. 1988), chimeric, humanized, in particular CDR-grafted antibodies, and dia or tetrabodies (Holliger et al. 1993). Also comprised are immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample. In this context the term “specific binding” refers to antibodies raised against the molecule of interest or a fragment thereof.
  • An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.
  • the anti-Adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is monospecific.
  • Monospecific anti-adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold means that said antibody or antibody fragment or non-Ig scaffold binds to one specific region encompassing at least 5 amino acids within the target ADM.
  • Monospecific anti-Adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold are anti-adrenomedullin (ADM) antibodies or anti-adrenomedullin antibody fragments or anti-ADM non-Ig scaffolds that all have affinity for the same antigen.
  • Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell.
  • Said anti-ADM antibody or antibody fragment binding to ADM or non-Ig scaffold binding to ADM may be a non-neutralizing anti-ADM antibody or antibody fragment binding to ADM or non-Ig scaffold binding to ADM.
  • said anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment or non-Ig scaffold.
  • a neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold would block the bioactivity of ADM to nearly 100%, to at least more than 90%, preferably to at least more than 95%.
  • a non-neutralizing anti-ADM antibody, or anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the bioactivity of ADM less than 100%, preferably to less than 95%, preferably to less than 90%, more preferred to less than 80% and even more preferred to less than 50%.
  • bioactivity of ADM is reduced to less than 100%, to 95% or less but not more, to 90% or less but not more, to 80% or less but not more, to 50% or less but not more
  • residual bioactivity of ADM bound to the non-neutralizing anti-ADM antibody, or anti-ADM antibody fragment or anti-ADM non-Ig scaffold would be more than 0%, preferably more than 5%, preferably more than 10%, more preferred more than 20%, more preferred more than 50%.
  • An antibody or fragment according to the present invention is a protein including one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG 1 , IgG 2 , IgG3, IgG 4 ), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length.
  • Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acids in length.
  • Light chains are encoded by a variable region gene at the NH 2 -terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus.
  • Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
  • the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3.
  • a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Pat. No. 5,807,715.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Pat. No. 5,585,089).
  • a human antibody is an antibody wherein the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest.
  • Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell.
  • Human antibodies can also be produced by phage display methods (see, e.g., WO91/17271; WO92/001047; WO92/20791) or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (for example, see WO93/12227; WO 91/10741).
  • the anti-ADM antibody may have the formats known in the art.
  • Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies.
  • antibodies according to the present invention are recombinantly produced antibodies as e.g. IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F-variable domain of heavy and/or light chain as e.g. chemically coupled antibodies (fragment antigen binding) including but not limited to Fab-fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g.
  • bivalent Fab-V5Sx2 bivalent Fab (mini-antibody) dimerized with the CH3 domain
  • bivalent Fab or multivalent Fab e.g. formed via multimerization with the aid of a heterologous domain, e.g. via dimerization of dHLX domains, e.g. Fab-dHLX-FSx2; F(ab′) 2 -fragments, scFv-fragments, multimerized multivalent or/and multi-specific scFv-fragments, bivalent and/or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional antibodies, polyvalent antibodies, e.g. from a different class than G; single-domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulins and numerous others.
  • biopolymer scaffolds are well known in the art to complex a target molecule and have been used for the generation of highly target specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins. For illustration of antibody formats please see FIGS. 1 a , 1 b and 1 c.
  • anti-ADM antibodies according to the present invention may be produced as outlined in Example 1 by synthesizing fragments of ADM as antigens. Thereafter, binder to said fragments are identified using the below described methods or other methods as known in the art.
  • Humanization of murine antibodies may be conducted according to the following procedure: For humanization of an antibody of murine origin the antibody sequence is analyzed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modelling (Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 2008 Jan. 1; 13:1619-33).
  • the ADM antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F(ab) 2 fragment and scFv-Fc Fusion protein.
  • the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFv fragment and bioavailability optimized conjugates thereof, such as PEGylated fragments.
  • One of the most preferred formats is scFab format.
  • the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a full-length antibody, antibody fragment, or non-Ig scaffold.
  • the anti-ADM antibody or an anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and can bind to an epitope of at least 5 amino acids in length contained in ADM.
  • the anti-ADM antibody or anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin is provided for use in therapy or prevention of shock in a patient, wherein said antibody or fragment or scaffold is not ADM-binding-Protein-1 (complement factor H).
  • the anti-Adrenomedullin (ADM) antibody or anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin is provided for use in therapy or prevention of shock in a patient, wherein said antibody or fragment or scaffold binds to a region of preferably at least 4, or at least 5 amino acids within the sequence of amino acid 1-21 of mature human ADM: YRQSMNNFQGLRSFGCRFGTC SEQ ID No.: 1
  • said anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold binds to a region or epitope of ADM that is located in the N-terminal part (amino acid 1-21) of adrenomedullin.
  • said anti-ADM-antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-14 of adrenomedullin: YRQSMNNFQGLRSF (SEQ ID No.: 15) that means to the N-terminal part (amino acid 1-14) of adrenomedullin.
  • said anti-ADM-antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-10 of adrenomedullin: YRQSMNNFQG (SEQ ID No.: 16); that means to the N-terminal part (amino acid 1-10) of adrenomedullin.
  • said anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-6 of adrenomedullin: YRQSMN (SEQ ID No.: 17); that means to the N-terminal part (amino acid 1-6) of adrenomedullin.
  • said region or epitope comprises preferably at least 4 or at least 5 amino acids in length.
  • said anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminal end (amino acid 1) of adrenomedullin.
  • N-terminal end means that the amino acid 1, that is “Y” of SEQ ID No. 13, or 18 (SEQ ID No.: 18 (1-42 of human ADM): YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA), respectively and is mandatory for binding.
  • the antibody or fragment or scaffold would neither bind N-terminal extended nor N-terminal modified Adrenomedullin nor N-terminal degraded adrenomedullin.
  • said anti-ADM-antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold binds only to a region within the sequence of mature ADM if the N-terminal end of ADM is free.
  • the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold would not bind to a region within the sequence of mature ADM if said sequence is e.g., comprised within pro-ADM.
  • N-terminal part (amino acid 1-21)” is understood by a person skilled in the art that the N-terminal part of ADM consists of amino acids 1-21 of the mature ADM sequence.
  • anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold does not bind to the C-terminal portion of ADM, i.e., the amino acid 43-52 of ADM:
  • An epitope also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies.
  • the epitope is the specific piece of the antigen to which an antibody binds.
  • the part of an antibody that binds to the epitope is called a paratope.
  • the epitopes of protein antigens are divided into two categories, conformational epitopes, and linear epitopes, based on their structure and interaction with the paratope.
  • Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen.
  • a conformational epitope is formed by the 3-D conformation adopted by the interaction of discontinuous amino acid residues.
  • a linear or a sequential epitope is an epitope that is recognized by antibodies by its linear sequence of amino acids, or primary structure and is formed by the 3-D conformation adopted by the interaction of contiguous amino acid residues.
  • an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to the present invention, wherein said anti-ADM antibody or said anti-ADM antibody fragment or anti-ADM non-Ig scaffold leads to an increase of the ADM level or ADM immunoreactivity in serum, blood, plasma of at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%.
  • an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to the present invention, wherein said anti-ADM antibody or said anti-ADM antibody fragment or anti-ADM non-Ig scaffold is an ADM stabilizing antibody or an ADM stabilizing antibody fragment or an ADM stabilizing non-Ig scaffold that enhances the half-life (t 1/2 ; half retention time) of adrenomedullin in serum, blood, plasma at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%.
  • the half-life (half retention time) of ADM may be determined in human serum, blood or plasma in absence and presence of an ADM stabilizing antibody or an ADM stabilizing antibody fragment or an ADM stabilizing non-Ig scaffold, respectively, using an immunoassay for the quantification of ADM.
  • the following steps may be conducted:
  • a two-fold increase of the half-life of ADM is an enhancement of half-life of 100%.
  • Example 3 An assay that may be used for the determination of the half-life (half retention time) of adrenomedullin in serum, blood, plasma is described in Example 3.
  • said anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment, or scaffold.
  • a neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold would block the bioactivity of ADM to nearly 100%, to at least more than 90%, preferably to at least more than 95%.
  • said non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the bioactivity of ADM to less than 100%, preferably less than 95% preferably less than 90%.
  • the non-neutralizing antibody is an antibody binding to a region of at least 5 amino acids within the sequence of amino acid 1-21 of mature human ADM (SEQ ID No.: 1).
  • a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or ADM non-Ig scaffold is used, wherein said anti-ADM antibody or an anti-ADM antibody fragment blocks the bioactivity of ADM to less than 80%, preferably less than 50% (of baseline values).
  • said limited blocking of the bioactivity (meaning reduction of the bioactivity) of ADM occurs even at excess concentration of the antibody, fragment or scaffold, meaning an excess of the antibody, fragment or scaffold in relation to ADM.
  • Said limited blocking is an intrinsic property of the ADM binder itself in said specific embodiment. This means that said antibody, fragment or scaffold has a maximal inhibition of 80% or 50% respectively.
  • said anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold would block the bioactivity/reduce the bioactivity of anti-ADM to at least 5%.
  • the stated above means that approximately 20% or 50% or even 95% residual ADM bioactivity remains present, respectively.
  • the provided anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM non-Ig scaffolds do not neutralize the respective ADM bioactivity.
  • a maximal inhibition in said ADM bioassay of 50% means that said anti-ADM antibody or said anti-ADM antibody fragment or said anti-ADM non-Ig scaffold, respectively, blocks the bioactivity of ADM to 50% of baseline values.
  • a maximal inhibition in said ADM bioassay of 80% means that said anti-ADM antibody or said anti-adrenomedullin antibody fragment or said anti-adrenomedullin non-Ig scaffold, respectively, blocks the bioactivity of ADM to 80%. This is in the sense of blocking the ADM bioactivity to not more than 80%. This means approximately 20% residual ADM bioactivity remains present.
  • the expression “blocks the bioactivity of ADM” in relation to the herein disclosed anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM non-Ig scaffolds should be understood as mere decreasing the bioactivity of ADM from 100% to 20% remaining ADM bioactivity at maximum, preferably decreasing the ADM bioactivity from 100% to 50% remaining ADM bioactivity; but in any case there is ADM bioactivity remaining that can be determined as detailed above.
  • a modulating anti-ADM antibody or a modulating anti-ADM antibody fragment or a modulating anti-ADM non-Ig scaffold is used in therapy or prevention of shock in a patient.
  • the present invention also relates to a pharmaceutical formulation comprising a therapeutically effective dose of the active ingredient, in combination with at least one pharmaceutically acceptable excipient.
  • the aqueous pharmaceutical composition can be administered to a subject in need of treatment, in accordance with known methods of administration.
  • methods of administration can further include shock dose injection or infusion over a certain period of time, intracerebrospinal, transdermal, oral, topical application or inhalation.
  • the volume of a dose in the formulation is about 20 ml, about 19 ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about 13 ml, about 12 ml, about 1 1 ml, about 10 ml, about 9 ml, about 8 ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml or about 1 ml.
  • the volume of the dose in a formulation is about 1 0.0 ml.
  • Dosage regimens may depend on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, in some embodiments, dosing from one-four times a week is contemplated. Even less frequent dosing may be used.
  • the dose is administered once every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 15 weeks, every 20 weeks, every 25 weeks, or longer.
  • the dose is administered once every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the dosing regimen can vary over time.
  • the dose of the formulation of the present invention is administered at an initial dose of 1-10 mg/kg body weight (e.g., intravenous injection), followed by 1-10 mg/kg body weight every other week starting one week after initial dose.
  • the appropriate dosage of the medicament will depend on the antibody employed, the type and severity of the disorder to be treated, whether the agent is administered for preventative or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician.
  • the clinician will administer the medicament, until a dosage is reached that achieves the desired result. Dosages may be determined empirically. For example, individuals are given incremental dosages to assess efficacy of the medicament, blood glucose levels may be followed.
  • Dose and/or frequency can vary over course of treatment. Empirical considerations, such as the antibody half-life, generally will contribute to the determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of one or more symptoms of autoimmune disease. In some individuals, more than one dose may be required. Frequency of administration may be determined and adjusted over the course of therapy. For example, without limitation, for repeated administrations over several days or longer, depending on the disease and its severity, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved.
  • Administration of the formulation of the present invention can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the formulation of the present invention can be essentially continuous over a preselected period of time or may be in a series of spaced dose.
  • the administration of the dose is a parenteral administration preferably selected from intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intra-ossial, intradermal and subcutaneous.
  • parenteral administration e.g., subcutaneous administration.
  • CDR1 SEQ ID NO: 2 GYTFSRYW
  • CDR2 SEQ ID NO: 3
  • CDR3 SEQ ID NO: 4 TEGYEYDGFDY
  • CDR1 SEQ ID NO: 5 QSIVYSNGNTY
  • CDR2 SEQ ID NO: 6 RVS
  • CDR3 SEQ ID NO: 7 FQGSHIPYT.
  • FIG. 1 A-C Relative area [%] of Main Peak (A), HMWS (B) and LMWS (C) as determined by SEC-HPLC of unstressed and stressed anti-ADM antibody (HAM8101) samples. Graphs show mean value of two replicates measured each two times.
  • FIG. 3 A-C Relative area [%] of Main Peak (A), Acidic Peaks (B) and Basic Peaks (C) as determined by CEX-HPLC of unstressed and stressed anti-ADM antibody (HAM8101) samples. Graphs show mean value of two replicates measured each two times.
  • FIG. 5 Results of DLS measurement of unstressed and stressed ant-ADM antibody (HAM8101) samples.
  • FIG. 6 A-B Results of non-reduced CE SDS after accelerated Aging (A), Mechanical stress (B) and Freeze and Thaw show mean value and SD of two replicates measured each two times.
  • FIG. 7 A-B Results of reduced CE SDS after accelerated Aging (A), Mechanical stress (B) and Freeze and Thaw show mean value and SD of two replicates measured each two times.
  • FIG. 8 DLS results of formulations 1-5 after storage at 40° C. and after mechanical stress.
  • FIG. 13 A-B Relative area [%] HMWS of SEC-HPLC of formulated anti-ADM antibody (HAM8101) stored for up to 2 weeks at 40° C. ⁇ 2° C./75% rH ⁇ 5% rH (A) and after mechanical stress and freeze & Thaw (B), graphs show mean and SD of two replicates measured each twice.
  • FIG. 16 A-D Subvisible Particle content below 5 ⁇ m (A), from 5 to 10 ⁇ m (B), above or equal to 10 ⁇ m (C) and above or equal to 25 ⁇ m (D) after mechanical stress.
  • FIG. 21 A-D Particles after thermal stress, Particle content below 5 ⁇ m (A), from 5 to 10 ⁇ m (B), above or equal to 10 ⁇ m (C) and above or equal to 25 ⁇ m (D) after 2 weeks of storage at 40° C.
  • HAM8101 All excipients used for the formulation of HAM8101 were sourced in Unites States Pharmacopoeia (USP) and/or European Pharmacopeia (Ph. Eur.) compliant quality. All chemicals used for analytical methods were sourced in a quality adequate for the individual method (e.g., p.a.). Drug substance (DS) samples of HAM8101 in basic buffer and current formulation were provided by the manufacturer. All samples were stored at ⁇ 80° C. with internal monitoring.
  • USP Unites States Pharmacopoeia
  • Ph. Eur. European Pharmacopeia
  • osmolality was performed by freezing point depression in an Osmometer (Osmomat 030-D RS, Gonotec). Each measurement was performed in 15 ⁇ l aliquots after calibration with sodium chloride standard (600 mOsmol/kg) and purified water.
  • the samples stored at 5 ⁇ 3° C. were kept in refrigerators with external temperature monitoring.
  • DS in original formulation was stored at ⁇ 80° C. in a freezer with internal monitoring of temperature.
  • CEX-HPLC method was based on the SOP provided by Rentschler for HAM8101.
  • the stored samples (20 ⁇ 2 mg/ml HAM8101) were separated on a MAbPac SCX-10 column (5 ⁇ m, 4 mm ⁇ 150 mm; Thermo Scientific, No.: 085198) on a Thermo Ultimate 3000 HPLC system.
  • a gradient (see Table 1) with Diluent A: 100% 20 mM 2-(N-morpholino) ethanesulfonic acid (MES) and Diluent B: 100% 20 mM MES with 200 mM sodium chloride with pH 6.0 over 40 minutes was used to separate the antibody charged variants at a constant flow rate of 0.5 ml/min at 25 ⁇ 5° C.
  • Per injection 60 ⁇ g DS diluted in Eluent A in 30 ⁇ l were loaded. Data were recorded at 280 nm and 214 nm by UV detector. Data analysis was performed with Chromeleon 7.2.6.10049 software.
  • DLS data were presented as D50-values over radius scatter plots of all measured peaks with intensity weighted symbol size in order to allow for easier comparison of the different formulations during storage time.
  • the radii for each population (x-axis) and the D50 value of all particles measured (y-axis) of each measurement are plotted within a log-log scatter plot.
  • each peak is proportional to the relative intensity of the respective peak (% Intensity weighted).
  • the overlay of replicate measurements using transparent color in addition enables the evaluation of the reproducibility of each signal by color intensity being useful in main peak determination.
  • each measurement consists of at least 2 ⁇ 100 single measurements. Parameters derive from a volume distribution and from an intensity distribution, which is itself obtained from a correlation function. Overall, the D50 value over peak radius plots with intensity weighted symbol size are used for qualitative comparison of the formulations at the applied time points within the different study arms of the stability study.
  • Turbidity of samples was analyzed by nephelometry or by measuring the absorption at 350 nm, 510 nm and 550 nm. Turbidity by absorption at 350 nm, 510 nm and 550 nm was measured using the Spark Plate Reader (Tecan, Sau). To do so, placebo and DS samples were measured in one 96 well plate under the same conditions.
  • turbidity of formulated drug substance was measured in cuvettes (HACH-Lange) with an inner diameter of 11 mm on a calibrated turbidimeter LAB 2100AN (HACH-Lange) by comparing the measured turbidity to standards with known turbidity ( ⁇ 0.1, 3, 6, 18, 20 and 30 NTU, acc. to European Pharmacopeia 6.0 as described in the StablCal®-Kit user manual; HACH-Lange).
  • Example 3 Design, Test and Optimization of Formulations to Stabilize an Anti-ADM Antibody (HAM8101) and Reduce Particle Formation in Liquid Formulation During Two Rounds of Accelerated Aging-Screening DoE Design
  • the purpose of this screening round was to investigate the influence of the different parameters of screening DoE, as shown in Table 5.
  • the reference of anti-ADM antibody (HAM8101) was formulated in current buffer (20 mM His/HCL, 300 mM Glycine, pH 6.0).
  • FIGS. 15 and 16 amount of Subvisible particles of samples was determined and the results are shown in FIGS. 15 and 16 .
  • Example 4 Design, Test and Optimization of Formulations to Stabilize an Anti-ADM Antibody (HAM8101) and Reduce Particle Formation in Liquid Formulation During Two Rounds of Accelerated Aging-Screening DoE Design-Optimization Round
  • Example 3 Based on the results of Example 3, 10 formulations were selected additional to the current formulation (Fit) and subjected to a second round of forced degradation. Stress conditions for Example 4 were identical to those used for Example 3. The composition of the different formulations is shown in Table 9.
  • Results of SEC-HPLC were expressed as mean relative peak area [%], derived from two replicates measured in duplicates and displayed in FIGS. 17 and 18 . Overall a pronounced fragmentation is detected after 4 weeks at 40° C. and an effect of rising HMW levels in Formulation 11 after 5 Freeze and Thaw cycles.
  • results of CE-SDS non-reduced (Table 12) and reduced (Table 13) were expressed as mean relative peak area derived from two replicates measured in duplicates and displayed in Table 12 and Table 13 respectively.
  • the results of CE-SDS (non-reduced) indicated that no distinct aggregation effect detectable, but a pronounced fragmentation was visible after 3.5 weeks at 40° C.
  • the lowest fragmentation level could be observed for F01 and F02, whereas the highest fragmentation level was observed for F08 and F11.
  • the results of CE-SDS (reduced) indicated that no distinct aggregation and fragmentation effect was detectable.
  • pronounced fragmentation was detectable after 3.5 weeks at 40° C. and the lowest fragmentation level was observed for F02, F03 and F09.
  • the DLS results after storage at 40° C. and after mechanical stress are depicted in FIG. 20 .

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